Colitis can be a life-threatening immune-related adverse event (irAE) for patients with metastatic melanoma treated with immune checkpoint blockade, a new anti-cancer immunotherapy. With the increasing use of PD-1/PD-L1 and CTLA-4 inhibitors, particularly in combination in melanoma and other cancers, timely and accurate diagnosis of colitis will become increasingly important for oncologists. The main goal of this study is to understand the clinical presentation of ipilimumab-induced colitis and to validate the use of CT scans as a safe and effective diagnostic tool. We analyzed a cohort of 303 patients who received ipilimumab at Dana Farber Cancer Institute on an expanded access protocol or standard of care between the years of 2008 and 2015. Age, number of doses and frequency of ipilimumab doses were found to be clinical characteristics which could help differentiate patients who develop ipilimumab induced colitis from those who only present with diarrhea and other gastrointestinal symptoms. Of the 303 patients, 100 (33%) developed diarrhea and 43 (14%) received treatment with corticosteroids for ipilimumab-induced colitis. For all patients with suspected immune-related colitis, an effort was made to firmly establish the diagnosis prior to or immediately after initiation of treatment. Forty-one of 43 patients (95%) who received steroids for presumed immune-related colitis had a colonoscopy and 27 of 43 (63%) patients had both computed tomography (CT) of the abdomen/pelvis and a colonoscopy including biopsy. In the 31 patients with a CT and biopsy, CT was highly predictive of the presence of colitis on biopsy (sensitivity 85%, specificity 75%, PPV 96%) and the absence of CT findings was predictive of a negative biopsy (negative LR 0.2). In the 44 patients who had symptoms and CT evaluation, CT was highly predictive of the need for steroids to reach resolution of symptoms (sensitivity 85%, specificity 88%, PPV 92%, positive LR 7.3). Fifteen of the 17 patients with negative CT findings did not require steroids to reach resolution of symptoms. In conclusion, CT of the abdomen/pelvis is a fast, reliable, and non-invasive mode of diagnosing ipilimumab-induced immune-related colitis, whereas colonoscopy may not be needed to firmly establish that diagnosis

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22371号 / 医博第4612号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 松田 文彦, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

No description available.

Final publication is available at http://www.bloodjournal.org/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19614号 / 医博第4121号 / 新制||医||1015(附属図書館) / 32650 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 山田 亮, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Large clinical trials testing hydrocortisone therapy in septic shock have produced conflicting results. Subgroups may benefit of hydrocortisone treatment depending on their individual immune response. We performed an exploratory analysis of the database from the international randomized controlled clinical trial Corticosteroid Therapy of Septic Shock (CORTICUS) employing machine learning to a panel of 137 variables collected from the Berlin subcohort comprising 83 patients including demographic and clinical measures, organ failure scores, leukocyte counts and levels of circulating cytokines. The identified theranostic marker was validated against data from a cohort of the Hellenic Sepsis Study Group (HSSG) (n = 246), patients enrolled in the clinical trial of Sodium Selenite and Procalcitonin Guided Antimicrobial Therapy in Severe Sepsis (SISPCT, n = 118), and another, smaller clinical trial (Crossover study, n = 20). In addition, in vitro blood culture experiments and in vivo experiments in mouse models were performed to assess biological plausibility. A low serum IFNg/IL10 ratio predicted increased survival in the hydrocortisone group whereas a high ratio predicted better survival in the placebo group. Using this marker for a decision rule, we applied it to three validation sets and observed the same trend. Experimental studies in vitro revealed that IFNg/IL10 was negatively associated with the load of (heat inactivated) pathogens in spiked human blood and in septic mouse models. Accordingly, an in silico analysis of published IFNg and IL10 values in bacteremic and non-bacteremic patients with the Systemic Inflammatory Response Syndrome supported this association between the ratio and pathogen burden. We propose IFNg/IL10 as a molecular marker supporting the decision to administer hydrocortisone to patients in septic shock. Prospective clinical studies are necessary and standard operating procedures need to be implemented, particularly to define a generic threshold. If confirmed, IFNg/IL10 may become a suitable theranostic marker for an urging clinical need.

Introduction: Immune therapy plays a larger role in cancer treatment these days, but in order to find new therapies and improve already existing ones, more knowledge about the immune system is needed. The peripheral blood contains many different cell types, some extensively studied, some less well-known. By using multicolor flow cytometry, the immune status of an individual can be displayed in relatively short time. Aim: The aim of this study was to develop a multicolor flow cytometry method in order to examine the distribution of 31 cell types, with a focus on immune regulatory cells, in peripheral blood in healthy donors, as well as examine the impact of cryopreservation on the different subsets. Methods: After isolating the mononuclear cells from peripheral blood using Ficoll separation, each sample (n = 19) were analyzed with three flow cytometry panels. The remaining cells were cryopreserved in -190°C and later thawed and analyzed the same way as the fresh samples were. Results: The cell distribution in fresh samples were mostly consistent with other studies. While the percentage of many cell types remained unchanged after thawing, the total percentage of T helper cells and some subsets were decreased in frozen samples, leading to a decrease in total T cells. Furthermore, the percentage of total monocytes were increased and the distribution of monocyte subsets were altered in frozen samples, among others. Conclusion: This study confirms the results of other studies of the human immune system and provides valuable knowledge about the impact of cryopreservation.

Des biomolécules telles que les protéines et les acides nucléiques sont d'un grand intérêt dans l'immunothérapie, la thérapie génique tels que des cancers, le traitement de SIDA, les troubles auto-immuns liés à la déficience immunitaire sévère et combinés à autres maladies. Afin de cibler ces objectifs, l'encapsulation et le ciblage sont incontestablement nécessaires pour protéger les biomolécules contre la dégradation rapide et aussi pour éviter les effets secondaires. Ainsi, dans le cadre de cette thèse, l'encapsulation de protéines et de l'ADN en utilisant le procédé basé sur la double émulsion évaporation de solvant a été étudiée. Mais avant de conduire l’étude expérimentale, l'état de l'art des procédés d'encapsulation a été effectué et une attention particulière a été consacrée à l’encapsulation de l'ADN et les protéines et aux procédés d’encapsulations basés sur la formulation des polymères biodégradables. En ce qui concerne l’étude expérimentale, une étude systématique des paramètres pertinents sur les propriétés physico-chimique et colloïdales des particules a été examinée. Cette étude a permis de mettre en évidence les facteurs clés capables d’affecter non seulement le procédé mais aussi les propriétés colloïdales des particules élaborées (taille, distribution en taille, propriétés électrocinétiques et stabilité colloïdale…). En second lieu, la formulation optimisée permettant l’obtention de tailles submicroniques a été utilisée pour l’encapsulation de l'ADN et les protéines. L'encapsulation de l'ADN a été étudiée en fonction de la concentration de l'ADN (sperme de saumon) et une encapsulation totale d'ADN a été observée sans modification ou altération des propriétés colloïdales des particules finales. L'encapsulation de protéines a été également examinée en utilisant HAS et une autre protéine fluorescente comme modèles. L'efficacité de l'encapsulation a été déterminée et trouvée de l’ordre de 100%. L'utilisation de la protéine fluorescente indique l'encapsulation totale avec une bonne répartition dans les cavités de la matrice polymère. En conclusion, le procédé de la double émulsion – évaporation de solvant est efficace pour l’encapsulation de l'ADN et des protéines sans modification des propriétés colloïdales des particules finales qui restent bien submicroniques / Biomolecules such as proteins and nucleic acids are of great interest in immunotherapy and gene therapy such as cancers, AIDS, autoimmune disorders, X-linked severe combined immune deficiency and many other diseases. In order to target such objectives, the encapsulation and the targeting are incontestably necessary principally to protect biomolecules against rapid degradation and also to avoid side effects. Then, in this thesis, the encapsulation of proteins and DNA using double emulsion solvent evaporation technique was performed. But before the encapsulation, the state of the art of the encapsulation processes was performed and special attention has been dedicated to DNA and proteins encapsulation. Firstly, systematic study of double emulsion solvent evaporation process was investigated in order to point out the key parameters controlling the particles size and the colloidal stability. Secondly, the optimized formulation conditions leading to submicron colloidal was used for DNA and Proteins encapsulation. The DNA encapsulation was investigated as a function of DNA concentration and it was found total encapsulation of DNA without any modification of the colloidal properties. The protein encapsulation was examined using HAS and fluorescent proteins as model. The encapsulation efficiency was found to be high and reaches 100%. The use of fluorescent protein shows the total encapsulation and good distribution of proteins in polymer matrix. As a general conclusion, the high encapsulation efficiency of DNA and protein, reveled the compatibility of the used process to encapsulated hydrophilic biomolecules even in submicron size biodegradable colloidal submicron particles

Les lymphocytes T régulateurs (Treg) jouent un rôle majeur dans la modulation de l’alloréactivité après allogreffe de cellules souches hématopoïétiques et permettent notamment de contrôler la réaction du greffon contre l’hôte (GVH) dans des modèles expérimentaux. Le potentiel thérapeutique des Treg est donc très important dans ce domaine, mais aussi dans l’auto-immunité ou en cancérologie. Cependant, de nombreuses barrières rendent difficile l’élaboration de stratégies thérapeutiques reposant sur le transfert adoptif de Treg chez l’homme et une meilleure compréhension des facteurs et mécanismes contrôlant la prolifération et les capacités suppressives de ces cellules permettrait de les cibler directement et si possible spécifiquement in vivo.Dans ce travail, après avoir élaboré un nouveau système d’évaluation clinique de la GVH chez la souris et démontré sa simplicité, sa reproductibilité et sa performance, nous avons pu montrer que l’action suppressive des Treg dans la GVH dépendait de l’interaction entre le TNFα produit par les lymphocytes T conventionnels (Tconv) du donneur et le récepteur TNFR2 exprimé par les Treg. En effet, en bloquant cette interaction de 3 façons différentes, à savoir par un anticorps monoclonal bloquant anti-TNFR2, ou en utilisant soit des Treg n’exprimant pas TNFR2 soit des Tconv ne produisant pas de TNFα, nous avons à chaque fois montré que l’effet protecteur des Treg était aboli en l’absence du signal TNF. Le récepteur TNFR2 étant exprimé préférentiellement par les Treg par rapport aux Tconv, nos résultats ouvrent la voie au ciblage des Treg in vivo via TNFR2, soit pour activer ce récepteur par un agoniste et donc stimuler les Treg afin de contrôler la GVH, soit à l’inverse pour bloquer l’axe TNFα/TNFR2 par un antagoniste et ainsi inhiber les Treg, ce qui permettrait alors de lever un frein à l’alloréactivité dans les situations où l’on cherche à la stimuler pour renforcer l’effet anti-tumoral, comme par exemple dans le cas des rechutes post-allogreffe. / Regulatory T cells (Tregs) are key players involved in the modulation of alloreactivity after hematopoietic stem cell transplantation. Indeed, Tregs can prevent graft-versus-host disease (GVHD) in experimental models. Therefore, the therapeutic potential of these cells in GVHD is substantial, as it is in other fields like auto-immunity or oncology. However, many obstacles still make the application of cellular therapy strategies based on the adoptive transfer of Tregs in humans quite complicated. A better understanding of factors and mechanisms that control the proliferation and suppressive capacities of Tregs could allow for a direct and specific targeting of these cells in vivo.In this work, after designing a new clinical grading system for murine GVHD and demonstrating its ease of use, reproducibility and performance, we have shown that the suppressive action of Tregs in GVHD depends on the interaction between TNFα produced by donor conventional T cells (Tconvs) and TNFR2 expressed by Tregs. Using 3 different ways to block this interaction, i.e. with an anti-TNFR2 blocking monoclonal antibody, or Tregs that do not express TNFR2 or donor Tconvs that cannot produce TNFα, we were able to show in each situation that blocking TNF signaling resulted in a loss of protection by Tregs. TNFR2 being highly expressed by Tregs as compared with Tconvs, our results pave the way for in vivo targeting of Tregs through TNFR2, either to activate this receptor with an agonist and therefore stimulate Tregs to control GVHD, or to block the TNFα/TNFR2 axis with an antagonist and in this case inhibit Tregs, which could boost alloreactivity, as expected in some particular settings like post-transplant relapse.